Peroxisomes are ubiquitous subcellular organelles intimately involved in multiple metabolic pathways in eukaryotic cells. The impairment of peroxisomal protein import and biogenesis is responsible for many human peroxisomal disorders (Zellweger's syndrome, Hyperpipecolic acidaemia, infantile Refsum's disease and neonatal adrenoleukodystrophy) which can be very debilitating and lethal. Our long term interest is in understanding the mechanism by which matrix and membrane proteins are transported from their site of synthesis, in the cytosol, to the peroxisomes. Two of the peroxisomal targeting signals, PTS1 and PTS2, involved in the import of peroxisomal matrix proteins, and the PTS1 receptor, have been identified and characterized. However, the other cytosolic and membrane components involved in peroxisomal protein import are unknown at present. We hope to use a combination of biochemical and genetic strategies to identify the other components involved in this import process. These studies will shed light on the biogenesis of an important subcellular organelle, and are also relevant to the human disorders mentioned above. The specific aims of the proposal are listed below. 1. To study the role of the P. pastoris PAS8 protein as the PTS1 receptor 2. Quantitation of peroxisomal import in vitro via the PTS1 pathway 3. Identification of yeast cytosolic factors required for peroxisomal import 4. Development of alternative cytosol-dependent in vitro import systems in yeast 5. To investigate the targeting and topology of peroxisomal membrane proteins 6. To study the role of the P. pastoris PAS1 and PAS5 proteins in peroxisome biogenesis